乙型肝炎病毒全基因组表观遗传修饰在核苷类似物停药复发中的作用机制研究

Viral relapse after nucleotide analogues(NA) withdrawal is one of the major concerns in the clinical management of chronic hepatitis B, however the mechanism remains unclear. Previous studies showed that viral epigenetic modification involved in the transcription regulation of HBV, and we speculate that viral epigenetic modification may be associated with the recurrence after NA withdrawal. The HBeAg positive chronic hepatitis B patients who received NA naive treatment and achieved cessation criterion are enrolled and liver biopsy is performed before drug withdrawal. The patients are then followed up prospectively. CHIP-Seq、WGBS、RT-PCR and western blot was applied to analyze the viral epigenetic modification including histone acetylation 、methylation and cccDNA methylation. The difference of status and position of epigenetic modification is compared between sustained response patients and viral relapse patients. The impact of NA treatment on the viral epigenetic modification is verified in the HBV replication cell model HepAD38, which treated by entecavir and then withdrawal and transfected with DNMT plasmid. The epigenetic modification changes during entecavir treatment and withdrawal are analyzed. The aim of this study is to investigate the impact of genome-wide HBV epigenetic modification changes on the cccDNA transcriptional activity during NA therapy in molecular、cellular and tissue level, to explore the molecular virological mechanism of recurrence after NA withdrawal and the novel target for preventing viral relapse, to provide novel insight for the safety cessation of NA therapy.

核苷类似物（NA）停药复发是慢性乙型肝炎（CHB）临床管理的热点和难点问题之一，其机制尚不明确。病毒表观遗传修饰参与了HBV的转录调控，推测可能与NA停药复发相关。本课题拟通过接受NA初治且达到停药标准CHB患者的肝组织，应用染色质免疫沉淀-高通量测序、全基因组甲基化测序、RT-PCR和Western Blot等技术，分析停药复发组和持久应答组在HBV组蛋白乙酰化、甲基化和cccDNA甲基化等表观遗传修饰状态和定位的差异；其次通过HBV复制细胞系HepAD38，予以恩替卡韦（ETV）处理，并在ETV撤药后转染DNMT质粒，从细胞模型上验证NA对HBV表观遗传修饰的影响；从HBV全基因组层次、从分子、细胞和组织水平研究NA抗病毒治疗后HBV表观遗传修饰变化对cccDNA转录活性的影响，探讨核苷类似物停药复发的分子病毒学机制，探索减少停药复发的干预靶点，为NA安全停药提供新的思路。

乙型肝炎病毒（HBV）的变异是影响慢性乙型肝炎疾病进展与疗效的重要因素，本项目主要从HBV全长基因组水平探讨病毒变异和表观遗传修饰对疾病表型和治疗的影响。主要研究内容包括：1. 分析慢性乙型肝炎免疫耐受期和免疫清除期患者HBV全长基因组准种特征，从准种水平探讨病毒变异与疾病表型的关联；2. 研究不同核苷类似物抗病毒药物对于HBV表观遗传修饰的影响，探讨影响抗病毒疗效的病毒因素；第一部分研究共入组90例免疫耐受期（IT）及200例慢性乙型病毒性肝炎（CHB）患者，通过HBV全基因组测序分析发现在BCP/PreC区的病毒准种特征与肝脏炎症及纤维化程度密切相关，聚类分析发现该区段的准种特征可以将患者显著分为IT和CHB两组。应用自主开发的QAP病毒准种分析软件，首次实现病毒准种的OTU定量。通过层次聚类（HCA）和主成分分析方法（PCA）将病毒准种定量与临床表型进行关联分析，发现病毒准种定量与肝纤维化及炎症等级密切相关。最后通过KNN、RF、SVM等机器学习方法建立IT患者的精准诊断模型，其鉴别IT或CHB患者的效能显著优于现有的HBsAg定量、APRI、FIB-4等模型。第二部分研究在体外实验观察恩替卡韦（ETV）和替诺福韦（TDF）对HBV表观遗传修饰的影响。在HepAD38细胞系中，未发现ETV或TDF对HBV甲基化的影响。相反地，应用甲基化芯片分析发现，ETV和TDF对HepAD38细胞DNA甲基化存在影响，且TDF干预组甲基化下调更为显著。进一步分析发现，TDF对细胞DNA甲基化转移酶DNMT存在浓度依赖性的抑制作用。通过慢病毒转染过表达或敲低DNMT3B，均未发现对HBV复制水平有影响。本项目首先基于病毒准种定量特征开发了一种全新的精准诊断模型，可以减少临床肝穿刺活检的需求，具有一定的临床应用前景。其次发现核苷类似物抗病毒治疗对宿主细胞DNA甲基化的影响，为解释不同抗病毒药物之间降低肝癌风险差异提供了新的思路。